1. Field of the Invention
The present invention relates to an immunoassay for quantitatively determining a constituent present in a very small amount in a sample, in which a reaction between an antigen and an antibody is utilized. More particularly, the present invention relates to a homogeneous immunoassay process for quantitatively determining an analyte antigen (ligand) in the sample by the use of an enzyme-labelled antibody, an enzyme-labelled antibody used therein, and a dry immunoassay element in which the homogeneous immunoassay of the invention is applied.
Analyses of the constituents originated from the living body or chemicals contained in the body fluids, such as blood or urine, are useful for diagnosing the condition of diseases or judging the course of curing, and thus they occupy important parts in the field of clinical test. The so-called enzyme immunoassay has been known in the art as one method for analyzing such constituents (ligands) generally present in a small amount in the body fluids. The enzyme immunoassay may be classified into a heterogeneous system for which B/F (Bound/Free) separation must be effected, and a homogeneous system in which B/F (Bound/Free) separation is not necessary. Meantime, B stands for the labelling material in a complex formed by binding of the specific antibody (or specific antigen) to the ligand, and F stands for the free labelling material which is not bound to the ligand.
In the heterogeneous system, the antigen-antibody bound (B) formed by the reaction between the antigen and the antibody is separated from free antibody and antigen (F) by any suitable means and then the activity of the labelling enzyme in the antigen-antibody bound is determined. Although it is expected that the heterogeneous system has a high sensitivity in principle since the bound (B) is separated from free antibody and antigen (F), there is a problem that cumbersome operations are needed for the B/F separation and thus a relatively long time is necessary for the determination.
On the other hand, the reactions in the homogeneous system are based on the phenomenon that the enzymatic activity of the labelling enzyme is affected by some interference caused by binding of an antibody to the antigen (ligand), and the inhibition due to antigen-antibody binding is generally utilized. In general, the antigen is labelled with an enzyme so that the suppression in enzymatic activity either by a steric hindrance imposed on binding of the enzyme, which is bound to a generally large molecule antibody, with the substrate or by a change in three-dimensional structure of the enzyme is detected. For example, EMIT (Enzyme Multiplied Immunoassay Technique) is well-known as such a system.
Alternatively, when the antigen is a high molecular weight substance, the antibody may be labelled with an enzyme and the suppression in enzymatic activity due to the antigen-antibody binding reaction may be utilized. The operations in the homogeneous systems are relatively simplified since complicated B/F separation is not necessary. However, the homogeneous system has a disadvantage that the sensitivity thereof is relatively low in principle.
2. Prior Art Statement
An improved homogeneous immunoassaying process has been disclosed in Unexamined Japanese Patent Publication No. 108756/1985 (corresponding to U.S. Pat. No. 4,692,404 and EP 0144176A). In this prior-proposed process, a water-insoluble high molecular weight polymer substrate is used as the substrate for the enzyme. The enzymatic reaction takes place on the surfaces of the substrate particles, in other words, at the solid-liquid interface. As the result, the suppression in enzymatic activity due to steric hindrance caused by binding between the enzyme-labelled antibody and the antigen is exaggerated. However, improvement in sensitivity by this prior-proposed process is limited, and there is a demand for a more sensitive immunoassay process.
In the routine clinical tests in which a number of test samples are to be handled, it is demanded that the individual samples should be analyzed rapidly by simple operations, more desirously by automated operation sequence. To comply with the demand, dry analysis elements have been proposed (for example, by Unexamined Japanese Patent Publication Nos. 53888/1974 (corresponding to U.S. Pat. No. 3,992,158), 77356/1984 (corresponding to EP 0097952A) and 102388/1984 (corresponding to U.S. Pat. No. 4,861,552) and U.S. Pat. No. 4,459,358. It is desirous that the immunoassay process can be applied to such a dry analysis element.